Arousal involves a state of heightened neural activity and lower threshold sensitivity to environmental stimuli. Substance abuse (SA) often involves the uncontrollable self- administration of drugs that alter levels of arousal. Epidemiological and genetic data suggest a linkage between SA, sleep disturbances and arousal/attentional disorders such as ADHD. Thus an understanding of the genetic and neural circuit-level mechanisms that control arousal may lend insight into the pathophysiology and genetics of SA. A fundamental question is whether arousal is a unitary, generalized state, or whether there are different forms of arousal, controlling different behaviors. Consistent with the latter view, multiple neuromodulatory systems have been implicated in arousal, including biogenic amines (BAs), acetylcholine (ACh), hormones and neuropeptides such as orexin. Furthermore, recent data from my laboratory have shown that in Drosophila, even a single neuromodulator (dopamine; DA) can influence different forms of arousal (sleep-wake vs. stress-induced) in opposite directions, by acting through different neural circuits. These data suggest that a knowledge of the specific neural substrates on which different neuromodulators act to influence arousal, in different behavioral settings, will be essential for understanding the circuitry of arousal-related disorders, including addiction. In this proposal, we will address the following questions, using Drosophila as a model system: 1) Does DA influence other forms of arousal, and if so through what circuits? 2) Which other neuromodulators influence arousal, what form(s) of arousal do they control and through which circuits do they act? To address these questions, we will develop a general method to visualize the activation of neuromodulator receptors on specific neural circuits by their endogenous ligands in vivo. We will use this method to visualize the neural circuits that are regulated by different neuromodulators in different behavioral paradigms that are influenced by arousal. If successful, this method should be broadly applicable to a variety of genetically accessible model organisms and could transform studies of neuromodulation and its role in SA, addiction, attentional and hyperactivity disorders, and sleep disturbances. PUBLIC HEALTH RELEVANCE: Neuromodulators such as dopamine play a key role in substance abuse, ADHD and sleep disorders. All of these disorders are linked to disturbances in arousal. Whether there are different forms of arousal, each linked to different behaviors and behavioral disorders, and the role of different neuromodulators in controlling various forms of arousal, is poorly understood. This proposal aims to develop new methodology to systematically map the roles of different neuromodulators in different settings of arousal.